1. PSI-2 Bottlenecks Meeting, April 14-16, 2008 A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION Cory Gerdts, Ph.D. April 15, 2008 PSI-2 Bottlenecks Meeting, Natcher Conference Center April 14-16, 2008 ATCG3D is a Specialized PSI-2 Center Funded by the NIGMS and NCRR, Grant # U54-GM074961-03 Slide prepared by Lance Stewart

2. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Outline  Technology Background  Project Update  Upcoming Improvements Slide prepared by Cory Gerdts

3. PSI-2 Bottlenecks Meeting, April 14-16, 2008 H. Song, J. D. Tice, R. F. Ismagilov Angew. Chem.-Int. Edit. 2003, 42, 768 Langmuir (2003) Appl. Phys. Lett. (2003) Anal. Chim. Acta (2004) Philos. T. Roy. Soc. A (2004) fluorocarbon Droplets (Plugs) in Microfluidic Channels Slide prepared by Cory Gerdts

4. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Labcard The Microfluidic Protein Crystallization System: (MPCS) Slide prepared by Cory Gerdts A Three Component System

5. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Crystallization in Plugs Slide prepared by Cory Gerdts

6. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Formation of Fine Gradients Zheng B.; Roach L. S.; R. F. Ismagilov J. Am. Chem. Soc. 2003, 125: 11170-11171. Zheng, B.; Ismagilov, R.F. Angew. Chem. 2005, 117: 2576-2579. Pre-formed Cartridge - ~10 nL plugs 200  m Gradient and Sparse Matrix Screening Sparse Matrix Screening with Pre-Formed Cartridges Slide prepared by Cory Gerdts

7. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Hybrid Method: Sparse Matrix + Gradient Screening + L. Li, D. Mustafi, Q. Fu, V. Tereshko, D.L. Chen, J.D. Tice, R.F. Ismagilov PNAS, 2006, 103, 19243. Slide prepared by Cory Gerdts

8. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Hybrid Method Slide prepared by Cory Gerdts

9. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Outline  Technology Background  Project Update  Upcoming Improvements Slide prepared by Cory Gerdts

10. PSI-2 Bottlenecks Meeting, April 14-16, 2008 MPCS Development Goals  Labcard  Flat  Thin  Rigid  Low Dead Volume Macro- Micro interface  Injection Molded Parts (low cost)  One-time Use  Plastic Material Properties:  Transparent  Low Birefringence  X-ray Transmissive  Low Surface Energy (hydrophobic/fluorophilic) Slide prepared by Cory Gerdts  Pump System  Constant Flow at Low Flow Rates  Ability to Form Smooth Gradients  No Delay/Response Time  Dissemination  Disseminate Technology to Center and Community  Make Technology Available

11. PSI-2 Bottlenecks Meeting, April 14-16, 2008 MPCS - Beta

12. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Multiple-Pronged Approach: 2 Functional Labcards Slide prepared by Cory Gerdts SBS Formatted Plate

13. PSI-2 Bottlenecks Meeting, April 14-16, 2008 MPCS Advantages/Features: Diffraction-Ready Crystals Slide prepared by Cory Gerdts Reaction Center Fatty Acid Amide Hydrolase Acyl-CoA hydrolase Porin Myoglobin TDP ribose-phosphate pyrophosphokinase methionine-R- sulfoxide reductase

14. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Protein Crystal Extraction Slide prepared by Cory Gerdts Methionine-R-sulfoxide Reductase data set: 1.7 Å resolution

15. PSI-2 Bottlenecks Meeting, April 14-16, 2008Slide prepared by Cory Gerdts MPCS Advantages/Features: In Situ Diffraction Data merged from 3 crystals: 1.9 Å resolution

16. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Improve efficiency: *Mutants* Ligands *DNA complexes pH 6pH 7 pH 8 * Cryoprotectants* Ligands * Other Protein Partners * Other DNA Partners * Detergents* Heavy Atoms Slide prepared by Cory Gerdts MPCS Advantages/Features: Optimization Applications of small volume fine gradient screening:  Optimize buffer system, precipitant concentration, and pH  Identify optimal or tolerable doses of additives to known crystallization conditions  Reduce protein preparatiion requirements for screening

17. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Results of a Smooth Gradient

18. PSI-2 Bottlenecks Meeting, April 14-16, 2008 4  L of protein is enough for ~800 experiments. As little as 0.5  L protein can be aspirated easily. Protein is aspirate into a piece of Teflon tubing on a syringe that is back-filled with the fluorocarbon (FC) oil. Every nL of protein gets displaced into the system. Slide prepared by Cory Gerdts MPCS Advantages/Features: No Dead Volume

19. PSI-2 Bottlenecks Meeting, April 14-16, 2008 3. The Labcard is filled with plugs that each contain one or a few small microcrystals. Microcrystals from a previous trial can be used to perform microfluidic seeding in the MPCS 1. Microcrystals are aspirated into a Teflon tube 2. The microcrystal-containing solution is used as one of the aqueous streams in the 3+1 mixer. Microfluidic seeding can be used to: Optimize a protein that nucleates too much, or Induce crystallization in a mutant, ligand, DNA, etc. screen by crushing up a crystal from a known condition or from the wild type version Slide prepared by Cory Gerdts MPCS Advantages/Features: Microfluidic Seeding

20. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Outline  Technology Background  Project Update  Upcoming Improvements Slide prepared by Cory Gerdts

21. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Microcapillary Crystallization: Next Steps  Development of more user friendly full instrument  Improved connections (hybrid in plastic Labcards)  Automatic Aspirator  Improved pumps  Further Dissemination

22. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Microcapillary Protein Crystallization Workshop Date: June 6-7, 2008 Location: deCODE biostructures Emerald BioSystems 2501 Davey Road Woodridge, IL 60517 Micropipet / Microcapillary Handling and Crystallization Counterdiffusion Microcapillary Crystallization Plug-Based Microcapillary Crystallization Slide prepared by Cory Gerdts and Lance Stewart

23. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Acknowledgements  Microcapillary Crystallization deCODE:  Lance Stewart (PI)  Peter Nollert  Mark Elliot  Yiping Xia The Scripps Research Institute:  Raymond Stevens (Co-PI)  Peter Kuhn  Peter Clark  Vadim Cherezov  Joe Ng  ATCG3D Funding NIGMS / NCRR U54-GM074961 University of Chicago:  Rustem Ismagilov  Liang Li  George Sawicki  Debarshi Mustafi  Valentina Terechko  Qiang Fu  Wenbin Du  Anna Selezneva For more information: Cory Gerdts Phone: 630-783-4691 Email: cgerdts@ emeraldbiosystems.com

24. PSI-2 Bottlenecks Meeting, April 14-16, 2008

25. Space group – P1 (Triclinic) Unit cell – a = 42.00, b = 45.17, c = 45.40 Angles –  = 88.4,  = 83.7,  = 69.1 Scaled from 50 – 1.7 Completeness – 95.3 (87.4) Rmerge – 6.8% (42.3) I/  I – 23.1 (2.2) # of reflections -118,181 (total), 32,539 (unique) Redundancy 3.6 (3.3) Methionine-R-sulfoxide Reductase data set

26. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Counterdiffusion Confined Geometry Microcapillary Crystallization  Topas COC plastic microfluidic crystallization Greiner Bio-One labcard  Individual Lysozyme Crystals grown in microcapillary channels by counterdiffision methods  In situ X-ray diffraction on individual crystals reveals different diffraction quality Slide prepared by Lance Stewart from data of Joe Ng and Peter Kuhn Greiner Bio-One Commercialization of Confined Geometry Counterdiffusion Labcard Prototype Prototype Design

27. PSI-2 Bottlenecks Meeting, April 14-16, 2008  USABLE soluble AND membrane protein crystals grow in ~10 nL plugs  In situ diffraction for both data acquisition and crystal quality screening  Increased efficiency from preparing decreasing amounts of protein  On-chip formulation for fine granularity screening for “narrow crystallization slots”  ~800 experiments per card  No dead volume – every nL gets used  Large Phase Space can be investigated with the Hybrid Method using preformed cartridges  Microfluidic seeding Summary of MPCS Advantages and Features Slide prepared by Cory Gerdts

28. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Nanovolume Microcapillary Crystallization with In Situ X-ray Imaging  Emerging Technologies  Microfluidic Device Production (PDMS, Plastic, Glass)  Microfluidic Pumping Systems (Various)  Novel Microfluidic Circuitry and Methods for Protein Crystallization (Seeding, Gradient, Random Sparse, Microbatch, Counterdiffusion)  Opportunity  Efficient Exploration of Crystallization Space per Unit Protein  Integration of Crystallization with X-ray Data Collection (In Situ)  Efficiency Gain  Increased Crystallization Success per Unit of Protein  Improved In situ Diffraction Quality Screening (true measure of a crystal)  Improved Inventory Staging of Crystals for X-ray Data Collection Slide prepared by Lance Stewart

29. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Simple 4-Microsyringe Pump System and “Microplugger” Control Software and Labcard Prototype pH Gradient Test, ~10nl Plugs Slide prepared by Lance Stewart Emerald BioSystems is offering commercial systems as of July 2007 In Situ X-ray Diffraction of Protein Crystals in Labcards Labcard Prototype Micro-Syringe Pump System Microplugger™ Control Software

30. PSI-2 Bottlenecks Meeting, April 14-16, 2008 ATCG3D Technology Focus Areas  Compact Light Source for Tunable X-ray Diffraction Data Collection in a Laboratory Setting  Nanovolume Microcapillary Crystallization with In Situ X-ray Imaging  Computer Aided Protein Construct Engineering and Synthetic Gene Design Slide prepared by Lance Stewart

31. PSI-2 Bottlenecks Meeting, April 14-16, 2008 DETECT-X Imaging of Protein Crystals in Microfluidic Devices  DETECT-X Imager used in conjunction with Millipol software  High signal to noise imaging of crystals  Crystal orientation imaging  Potential to Enable Efficient Complete in situ X-ray diffraction Data Set Collection Slide prepared by Lance Stewart with data from Peter Nollert W. Kaminski, Millipol Software Norgren Systems LLC Hardware Engineering Emerald BioSystems, Commercial Launch of DETECT-X, Q2 2007

32. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Future Directions Gene-to-3D in 3 Days ?  Starting from Oligonucleotides  PCR based gene production / transcription  mRNA, 8 h  Cell free translation for protein production,16 h  Affinity purification of protein,4 h  Nanovolume microcapillary labcard crystallization,20 h  In situ X-ray diffraction data collection,6 h  Structure Determination16 h  TOTAL ~70 h Slide prepared by Lance Stewart 3D in 3 Day Challenge

33. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Acknowledgements  Synthetic Gene Design Software  John Walchli (deCODE)  Alex Burgin (deCODE)  Mark Mixon (deCODE)  Don Lorimer (deCODE)  ATCG3D PI Collaborators  Peter Kuhn and Raymond Stevens (Co-PIs, The Scripps Research Institute) Slide prepared by Lance Stewart  Corporate Collaborators  Peter Nollert, Emerald BioSystems  Lawrence Kuo, J&JPRD  Masaki Madono, Cell Free Sciences  PSI-2 Grant Funding  ATCG3D Funding NIGMS / NCRR U54-GM074961  PSI-2 / SG Collaborators  James Love, NYCOMPS  Dmitriy Vinarov, CESG, Univ. of Wisconsin  Mark Sullivan, University of Rochester  Alexei Brooun, TSRI now at Pfizer  Yaeta Endo, Ehime University  Synthetic Gene Design Wet Lab Work  Don Lorimer (deCODE)  Ellen Wallace (deCODE)  Amy Raymond (deCODE)  Adrienne Metz (deCODE)  Rena Grice (deCODE)  SSGCID (NIAID) Collaborators  Peter Myler, Seattle Biomedical Research Institute  Wes Van Voorhis, University of Washington  Contract Funding  SSGCID Funding from NIAID HHSN272200700057C

34. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Microcapillary Protein Crystallization Workshop Date: June 6-7, 2008 Location: deCODE biostructures /Emerald BioSystems 2501 Davey Road Woodridge, IL 60517 Micropipet / Microcapillary Handling and Crystallization Counterdiffusion Microcapillary Crystallization Plug-Based Microcapillary Crystallization Slide prepared by Cory Gerdts www.MPCW2008.org

35. PSI-2 Bottlenecks Meeting, April 14-16, 2008

36. Storage capillary controls evaporation Membrane protein crystallization is done using Teflon capillaries and PDMS 3+1 mixers. Slide prepared by Cory Gerdts MPCS Advantages/Features: Membrane Proteins

37. PSI-2 Bottlenecks Meeting, April 14-16, 2008 ATCG3D

38. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Becoming a Beta User of the Microcapillary Protein Crystallization System  Beta User obtains complete Microcapillary Protein Crystallization System  Training: #1: training of Beta User scientists at Emerald site (Bainbridge Island, WA)  Installation: complete installation of the Microcapillary Protein Crystallization System in Beta User laboratory, validation and QC  Training #2: training of Beta User scientists at Beta User site  Cost: $24k for system, $19k for annual technology access license  Emerald solicits feedback to improve system performance  Beta User agrees not to reverse engineer system, resulting IP owned by Emerald, co-authorship of publication

39. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Nanovolume Protein Crystallization: PDMS 3+1 Mixer and Microcapillary Holder PDMS 3+1 Mixer 0.45 meter of Teflon capillary stored in glass capillary 1 meter of tubing, ~1000 membrane protein crystalliization trials Li, L., Mustafi, D., Fu, Q., Tereshko, V., Chen, D.L., Tice, J.D., Ismagilov, R.F. PNAS 2006, 103: 19243 Slide prepared by Cory Gerdts

40. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Crystallization of Oligoendopeptidase F by Microfluidic Seeding Microfluidic Separation of Nucleation and Crystal Growth Yields Single Crystals Target: apc36224 selected by MCSG but unsolved Only Precipitation and Crystal Clusters in Optimized Vapor Diffusion Experiments

41. PSI-2 Bottlenecks Meeting, April 14-16, 2008 Oligoendopeptidase F (B. stearothermophilus) Structure  Oligoendopeptidase F  apc36224  3.1 Å resolution  Space group: P3 1 2 1 ; Unit Cell  Parameters a=b 119.50 c=248.90  R-factor = 0.196, R-free = 0.248  Solvent Content: ~70%;  PDB Accession #s 2H1N and 2H1J  Zn ++ Metallopeptidase family M3  Structural similarity to:  human testicular Angiotensin-converting enzyme, 1O86  E.coli Dipeptidyl Carboxypeptidase Dcp, 1Y79  Neurolysin, 1I1I  Pyrococcus furiosus carboxypeptidase, 1K9X  ATCG3D and MCSG collaboration Gerdts, C.J., Tereshko, V., Yadav, M.K., Dementieva, I., Collart, F., Joachimiak, A., Stevens, R.C., Kuhn, P., Kossiakoff, A., and Ismagilov, R.F. Angew. Chem. Int. Ed. 2006 45: 8156-8160 Time-Controlled Microfluidic Seeding in nL-Volume Droplets To Separate Nucleation and Growth Stages of Protein Crystallization